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EP3617321B1 - Kit zur nukleinsäureextraktion und -verstärkung aus einer probe - Google Patents

Kit zur nukleinsäureextraktion und -verstärkung aus einer probe Download PDF

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Publication number
EP3617321B1
EP3617321B1 EP19183631.1A EP19183631A EP3617321B1 EP 3617321 B1 EP3617321 B1 EP 3617321B1 EP 19183631 A EP19183631 A EP 19183631A EP 3617321 B1 EP3617321 B1 EP 3617321B1
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Prior art keywords
nucleic acid
dna
coated
kit
target nucleic
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French (fr)
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EP3617321A3 (de
EP3617321A2 (de
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Carolyn R. Hoyal-Wrightson
Andreas Braun
Karsten E. Schmidt
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Sequenom Inc
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Sequenom Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • kits as characterized in the claims.
  • the methods as also described herein may be used in a wide range of applications, including the extraction of fetal nucleic acids from maternal plasma, the detection of circulating nucleic acids from neoplasms (malignant or non-malignant), the detection of early onset of tissue rejection, or any other application requiring the selective separation of nucleic acids based on their size and/or apoptotic origin.
  • US 5,234,809 relates to a process, a combination of means for isolating nucleic acid from a nucleic acid-containing starting material and a testkit in order to amplify the nucleic acid obtained by said process.
  • Isolated, purified nucleic acids may be used, inter alia, as a starting material for diagnosis and prognosis of diseases or disorders. Therefore, the isolation of nucleic acids, particularly by non-invasive means, is of particular importance for use in genetic analyses.
  • Chromatography-based methods increase flexibility and automation since these methods can be used in combination with multiple matrices (e.g. , membranes, latex, magnetic beads, micro-titer plate, etc.) and in the presence or absence of ligands (e.g. , DEAE, silica, acrylamide, etc.). However, these methods are better suited to extract larger strands of nucleic acids to ensure greater success in downstream analysis.
  • multiple matrices e.g. , membranes, latex, magnetic beads, micro-titer plate, etc.
  • ligands e.g. , DEAE, silica, acrylamide, etc.
  • the invention relates to a kit for extracting target nucleic acid comprising: a) an aqueous solution of functional group-coated paramagnetic microparticles; b) a first binding buffer formulated to comprise a suitable salt, wherein the salt is present at a concentration appropriate for binding a non-target nucleic acid of 1200 or more base pairs to the paramagnetic microparticles; and c) a second binding buffer formulated to comprise a suitable salt, wherein the salt is present at a higher concentration than that of the first binding buffer, appropriate for binding a target nucleic acid less than 1200 base pairs, to the paramagnetic microparticles, wherein the suitable salt in the first binding buffer and in the second binding buffer is at least one compound selected from the group consisting of guanidine salt, sodium io
  • the kit of the invention comprises reagents for the formulation of a wash buffer and an elution buffer, wherein the wash buffer dissolves impurities, but not the nucleic acid bound to paramagnetic microparticles and the elution buffer is a non-salt buffered solution with a pH range between about 7.0 to about 8.5.
  • the functional group-coated paramagnetic microparticles are silica-coated, hydroxyl-coated, amine-coated, carboxyl-coated or encapsulated carboxyl group-coated.
  • the salt is guanidine (iso)thiocyanate or sodium perchlorate.
  • cell-free nucleic acid may originate from several sources, it has been demonstrated that one source of circulating extracellular nucleic acid originates from programmed cell death, also known as apoptosis.
  • the source of nucleic acid that arise as a result of apoptosis may be found in many body fluids and originate from several sources, including, but not limited to, normal programmed cell death in the host, induced programmed cell death in the case of an autoimmune disease, septic shock, neoplasms (malignant or non-malignant), or non-host sources such as an allograft (transplanted tissue), or the fetus or placenta of a pregnant woman.
  • the applications for the detection, extraction and relative enrichment of extracellular nucleic acid from peripheral blood or other body fluids are widespread and may include inter alia, non-invasive prenatal diagnosis, cancer diagnostics, pathogen detection, auto-immune response and allograft rejection.
  • the present disclosure includes methods, compositions and kits to extract and relatively enrich by physical separation or amplification short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids). More specifically, the present disclosure provides compositions, methods and kits for the selective extraction and relative enrichment, based on size discrimination, of nucleic acid of approximately 1,200 base pairs or less (herein referred to as "target nucleic acid”) in a high background of genomic nucleic acids (herein referred to as "non-target nucleic acid”). This leads to a relatively enriched fraction of nucleic acid that has a higher concentration of smaller nucleic acids.
  • target nucleic acid a high background of genomic nucleic acids
  • the methods of the present disclosure may be used to improve pathogen detection.
  • Methods for rapid identification of unknown bioagents using a combination of nucleic acid amplification and determination of base composition of informative amplicons by molecular mass analysis are disclosed in published U.S. Patent applications 20030027135 , 20030082539 , 20030124556 , 20030175696 , 20030175695 , 20030175697 , and 20030190605 and U.S. patent application 2003190605 , 2008209971 , 2004219517 and 2004180328 .
  • host cell as used herein is any cell into which exogenous nucleic acid can be introduced, producing a host cell which contains exogenous nucleic acid, in addition to host cell nucleic acid.
  • host cell nucleic acid and “endogenous nucleic acid” refer to nucleic acid species (e.g., genomic or chromosomal nucleic acid) that are present in a host cell as the cell is obtained.
  • exogenous refers to nucleic acid other than host cell nucleic acid; exogenous nucleic acid can be present into a host cell as a result of being introduced in the host cell or being introduced into an ancestor of the host cell.
  • a nucleic acid species which is exogenous to a particular host cell is a nucleic acid species which is non-endogenous (not present in the host cell as it was obtained or an ancestor of the host cell).
  • Appropriate host cells include, but are not limited to, bacterial cells, yeast cells, plant cells and mammalian cells.
  • extraction refers to the partial or complete separation and isolation of a nucleic acid from a biological or non-biological sample comprising other nucleic acids.
  • selective and selective refer to the ability to extract a particular species of nucleic acid molecule, on the basis of molecular size from a combination which includes or is a mixture of species of nucleic acid molecules.
  • nucleic acid and “nucleic acid molecule” may be used interchangeably throughout the disclosure.
  • the terms refer to a deoxyribonucleotide (DNA), ribonucleotide polymer (RNA), RNA/DNA hybrids and polyamide nucleic acids (PNAs) in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • DNA deoxyribonucleotide
  • RNA ribonucleotide polymer
  • PNAs polyamide nucleic acids
  • target nucleic acid refers to the nucleic acid of interest that is extracted based on its molecular size, preferably in a second extraction step, and further isolated for downstream analysis.
  • the target nucleic acid may have a molecular size smaller than the non-target nucleic acid present in the biological sample, for example, smaller than 1200 base pairs.
  • the target nucleic acid may be from apoptotic DNA, fetal DNA, oncogenic DNA, or any non-host DNA.
  • the target nucleic acid may be cell-free nucleic acid.
  • the target nucleic acid may be oligonucleosomal nucleic acid generated during programmed cell death.
  • non-target nucleic acid refers to the relatively high amount of non-desired background nucleic acid present in a biological sample, which is extracted, preferably, in a first extraction step.
  • Non-target nucleic acid may have a molecular size larger than target nucleic acid, for example, greater than 1200 base pairs.
  • Non-target nucleic acid may be from a host or host cell.
  • Non-target nucleic acid may be of maternal origin.
  • molecular size refers to the size of a nucleic acid molecule, which may be measured in terms of a nucleic acid molecule's mass or length (bases or base pairs).
  • Fetal nucleic acid is present in maternal plasma from the first trimester onwards, with concentrations that increase with progressing gestational age ( Lo et al. Am J Hum Genet (1998) 62:768-775 ). After delivery, fetal nucleic acid is cleared very rapidly from the maternal plasma ( Lo et al. Am J Hum Genet (1999) 64:218-224 ). Fetal nucleic acid is present in maternal plasma in a much higher fractional concentration than fetal nucleic acid in the cellular fraction of maternal blood ( Lo et al. Am J Hum Genet (1998) 62:768-775 ).
  • the target nucleic acid may be of fetal origin
  • the non-target nucleic acid may be of maternal origin.
  • the present disclosure relates to extracting nucleic acid from a biological sample such as whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinal fluid, lavage fluid (e.g ., bronchoalveolar, gastric, peritoneal, ductal, ear, athroscopic), biopsy sample, urine, feces, sputum, saliva, nasal mucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells.
  • the biological sample may be blood, and more preferably plasma.
  • blood encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined.
  • Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants.
  • Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated.
  • blood handling protocols are followed to ensure minimal degradation of nucleic acid in the sample and to minimize the creation of apoptotic nucleic acid in the sample. Blood handling methods are well known in the art.
  • the biological sample may be cell-free or substantially cell-free.
  • the biological sample may be a sample containing previously extracted, isolated or purified nucleic acids.
  • One way of targeting target nucleic acid is to use the non-cellular fraction of a biological sample; thus limiting the amount of intact cellular material (e.g. , large strand genomic DNA) from contaminating the sample.
  • a cell-free sample such as pre-cleared plasma, urine, etc. may be first treated to inactivate intracellular nucleases through the addition of an enzyme, a chaotropic substance, a detergent or any combination thereof.
  • the biological sample may be first treated to remove substantially all cells from the sample by any of the methods known in the art, for example, centrifugation, filtration, affinity chromatography, etc.
  • concentration sufficient to selectively bind refers to an amount sufficient to cause at least 50%, more preferably 70%, even more preferably 90% or more of the target nucleic acid to bind to an adsorptive surface.
  • Suitable solid phase carriers include, but are not limited to, other particles, fibers, beads and or supports which have an affinity for nucleic acids or may be modified ( e.g. , the addition of a functional group or groups) to bind nucleic acids, and which can embody a variety of shapes, that are either regular or irregular in form, provided that the shape maximizes the surface area of the solid phase, and embodies a carrier which is amenable to microscale manipulations. Silica-coated magnetic beads may be used.
  • the solid support may be modified to reversibly bind nucleic acid.
  • the solid support may have a functional group-coated surface.
  • the functional group-coated surface may be silica-coated, hydroxyl-coated, amine-coated, carboxyl-coated and encapsulated carboxyl group-coated.
  • the term "functional group-coated surface” as used herein refers to a surface which is coated with moieties which reversibly bind nucleic acids.
  • One example is a surface which is coated with moieties which each have a free functional group which is bound to the amino group of the amino silane or the solid support; as a result, the surfaces of the solid support are coated with the functional group containing moieties.
  • the functional group may be a carboxylic acid.
  • a suitable moiety with a free carboxylic acid functional group is a succinic acid moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the paramagnetic microparticle.
  • Suitable solid phase carriers having a functional group coated surface that reversibly binds nucleic acid molecules are for example, magnetically responsive solid phase carriers having a functional group-coated surface, such as, but not limited to, silica-coated, hydroxyl-coated, amino-coated, carboxyl-coated and encapsulated carboxyl group-coated magnetic beads.
  • an oligonucleotide e.g. , an adapter or primer
  • biotin which may bind to immobilized streptavidin.
  • nucleic acid from biological material requires cell lysis, inactivation of cellular nucleases and separation of the desired nucleic acid from cellular debris.
  • Common lysis procedures include mechanical disruption (e.g. , grinding, hypotonic lysis), chemical treatment (e.g. , detergent lysis, chaotropic agents, thiol reduction), and enzymatic digestion (e.g. , proteinase K).
  • the biological sample may be first lysed in the presence of a lysis buffer, chaotropic agent (e.g. , salt) and proteinase or protease.
  • chaotropic agent e.g. , salt
  • a single solution may contain detergents to solubilise cell membranes and strong chaotropic salts to inactivate intracellular enzymes. After cell lysis and nuclease inactivation, cellular debris may easily be removed by filtration or precipitation.
  • Lysis may be blocked.
  • the sample may be mixed with an agent that inhibits cell lysis to inhibit the lysis of cells, if cells are present, where the agent is a membrane stabilizer, a cross-linker, or a cell lysis inhibitor.
  • the agent may be a cell lysis inhibitor, and may be glutaraldehyde, derivatives of glutaraldehyde, formaldehyde, formalin, or derivatives of formaldehyde. See U.S. patent application 20040137470 .
  • the method may include adding a washing step or steps to remove non-nucleic acid molecules, for example salts, from the solid-support-target nucleic acid complex or surrounding solution.
  • Non-nucleic acid molecules are then removed with an alcohol-based wash and the target nucleic acid is eluted under low- or no-salt conditions (TE buffer or water) in small volumes, ready for immediate use without further concentration.
  • Extraction may be improved by the introduction of a carrier such as tRNA, glycogen, polyA RNA, dextran blue, linear poly acrylamide (LPA), or any material that increases the recovery of nucleic acid.
  • the carriers may be added to the second binding solution or washing buffer.
  • the final relative percentage of target nucleic acid to non-target nucleic acid may be at least about 5-6% fetal DNA, about 7-8% fetal DNA, about 9-10% fetal DNA, about 11-12% fetal DNA, about 13-14% fetal DNA.
  • fetal DNA about 15-16% fetal DNA, about 16-17% fetal DNA, about 17-18% fetal DNA, about 18-19% fetal DNA, about 19-20% fetal DNA, about 20-21% fetal DNA, about 21-22% fetal DNA, about 22-23% fetal DNA, about 23-24% fetal DNA, about 24-25% fetal DNA, about 25-35% fetal DNA, about 35-45% fetal DNA, about 45-55% fetal DNA, about 55-65% fetal DNA, about 65-75% fetal DNA, about 75-85% fetal DNA, about 85-90% fetal DNA, about 90-91% fetal DNA, about 91-92% fetal DNA, about 92-93% fetal DNA, about 93-94% fetal DNA, about 94-95% fetal DNA, about 95-96% fetal DNA, about 96-97% fetal DNA, about 97-98% fetal DNA, about 98-99% fetal DNA, or about 99-99.7% fetal
  • the methods provided herein may also be modified to combine steps, for example, in order to improve automation.
  • the methods of the present disclosure may be used in conjunction with any known technique suitable for the extraction, isolation or purification of nucleic acids, including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon 100 filter, Chemagen viral DNA/RNA 1k kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QlAfilter plasmid kit, Promega DNA purification systems, MangeSil Paramagnetic Particle based systems, Wizard SV technology, Wizard Genomic DNA purification kit, Amersham purification systems, GFX Genomic Blood DNA purification kit, Invitrogen Life Technologies Purification Systems, CONCERT purification system, Mo Bio Laboratories purification systems, UltraClean BloodSpin Kits, and UlraClean Blood DNA Kit.
  • cesium chloride gradients including cesium chloride gradients, gradients, sucrose gradients, glucose
  • the first extraction method may be any known or modified technique suitable for the extraction, isolation or purification of non-target nucleic acids (i.e ., larger than target nucleic acids), including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon 100 filter, Chemagen viral DNA/RNA 1k kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QlAfilter plasmid kit, Promega DNA purification systems, MangeSil Paramagnetic Particle based systems, Wizard SV technology, Wizard Genomic DNA purification kit, Amersham purification systems, GFX Genomic Blood DNA purification kit, Invitrogen Life Technologies Purification Systems, CONCERT purification system, Mo Bio Laboratories purification systems, UltraClean BloodSpin Kits, and UlraClean Blood DNA Kit.
  • One or more of the above methods may be modified
  • the second extraction method may be any known or modified technique suitable for the extraction, isolation or purification of target nucleic acids (i.e ., smaller than non-target nucleic acids), including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon 100 filter, Chemagen viral DNA/RNA 1k kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QlAfilter plasmid kit, Promega DNA purification systems, MangeSil Paramagnetic Particle based systems, Wizard SV technology, Wizard Genomic DNA purification kit, Amersham purification systems, GFX Genomic Blood DNA purification kit, Invitrogen Life Technologies Purification Systems, CONCERT purification system, Mo Bio Laboratories purification systems, UltraClean BloodSpin Kits, and UlraClean Blood DNA Kit.
  • One or more of the above methods may be modified
  • the present specification also further relates to kits for practicing the disclosed methods.
  • apoptosis is an essential mechanism in morphogenesis, development, differentiation, and homeostasis in all multicellular organisms.
  • apoptosis is distinguished from necrosis by activation of specific pathways that result in characteristic morphological features including DNA fragmentation, chromatin condensation, cytoplasmic and nuclear breakdown, and the formation of apoptotic bodies.
  • CAD Caspase-activated DNase
  • DFF DNA fragmentation factor
  • any method that can select for DNA fragments with blunt, 5'- phosphorylated ends is suitable to select for specific features (such as size, sequence and DNA base methylation differences) of the apoptotic DNA in a given biological sample. See for example, US patent applications 20050019769 , 20050164241 , 20030044388 , or 20060019278 .
  • a method For enrichment and detection of apoptotic DNA ladders in mammalian tissues, a method has been described that takes advantage of the presence of blunt, 5'-phosphorylated ends in apoptotic DNA by ligation of synthetic, blunt-ended linkers to both ends of linear apoptotic DNA fragments with T4 ligase which is able to form a covalent bond between the 3'-hydroxy ends of the synthetic linker and the 5'-phosphorylated ends of the DNA fragments ( Staley et al, Cell Death Differ. 1997 Jan;4(1):66-75 ).
  • the method can only be used as a generic tool to characterize the size distribution of apoptotic ladders in specific tissues in general, and is not site or sequence specific.
  • the present specification therefore, also provides a method for selectively amplifying short, fragmented nucleic acid by adapter mediated ligation and other related methods.
  • the method capitalizes on the blunt end and 5'-phosphorylated nature of the target nucleic acid as a means to attach a non-genome specific adapter to the blunt ends using a ligation process. While the nature of the termini of all cell-free nucleic acid is unknown, coupling this method with short extension times during amplification will favor the amplification of the oligonucleosome monomer and short multimers. Since the target nucleic acid is shorter than the non-targeted nucleic acid, the target nucleic acid can be enriched over the non-target nucleic acid.
  • This method can be further coupled with specific amplification of a nucleic acid region of interest for further analysis.
  • the 3' and 5' dephosphorylated adapters are complementary and form a double-stranded blunt end adapter complex.
  • the 5' adapter of the adapter complex ligates to the 5' phosphorylated strand of the target nucleic acid, and heat is introduced to release the shorter, unligated 3' adapter.
  • the 5' protruding ends of the ligated complex are filled in by a thermostable DNA polymerase.
  • the 5' adapter is reintroduced and serves as a PCR primer for whole genome amplification.
  • Oligonucleosomes are the repeating structural units of chromatin, each consisting of approximately 200 base pairs of DNA wound around a histone core that partially protects the DNA from nuclease digestion in vitro and in vivo. These units can be found as monomers or multimers and produce what is commonly referred to as an apoptotic DNA ladder. The units are formed by nuclease digestion of the flanking DNA not bound to histone resulting in the majority of oligonucleosomes being blunt ended and 5'-phorsphorylated.
  • oligonucleosomes are hard to detect and harder to isolate; however, they can serve as predictors for disease and other conditions (see US patent application 20040009518 ).
  • 5' dephosphorylated adapter refers to a nucleic acid which comprises about 20 to 30 base pairs that is complementary to a short dephosphorylated adapter and capable of hybridizing thereto to form a double-stranded, blunt end adapter complex capable of ligating to target nucleic acid. Specifically, the 5'adapter ligates to the 5'phosphorylated base of the target nucleic acid.
  • 3' dephosphorylated adapter refers to a nucleic acid which comprises about 10 to 15 base pairs that is complementary to the 5' adapter at the 3' end, thus capable of creating a double-stranded blunted end necessary for ligation.
  • the 3' adapter does not bind or ligate to the oligonucleosomal DNA.
  • 5' adapter primer refers to the same oligonucleotide sequence as the 5' dephosphorylated adapter, but is later reintroduced to the ligated sample to facilitate the whole genome amplification.
  • adapter complex refers to the hybridized, double-stranded 5' adapter and 3' adapter molecule.
  • the method is semi quantitative. By comparing the numbers of PCR cycles needed to detect target nucleic acid in two samples, the relative amount of target nucleic acid occurring in each sample can be estimated.
  • the 5' adapters may be bound to a solid support for increased enrichment of the target nucleic acid.
  • non-ligated, non-target nucleic acid is substantially removed from the solution, and amplification can proceed using only the targeted material that has ligated to the 5' adapters. This improves the enrichment of the target nucleic acid by removing genomic non-target nucleic acid that may compete with the target nucleic acid in the target-specific amplification step.
  • the target sequence is present in both the mother and the fetus, and the maternal sample is very abundant (>95%), under normal circumstances the fetal nucleic acid would not be detectable as it would be out-competed by the maternal nucleic acid in the first cycles of amplification.
  • the fetal nucleic acid is part or wholly oligonucleosomal in nature, and the majority of the ligated sample is fetal in nature, maternal nucleic acid is still present in the sample, which can compete with the fetal nucleic acid in the target-specific amplification step. Separation of non-target nucleic acid from the target nucleic acid (i.e.
  • ligated sample increases the detection of fetal nucleic acid in cases where there is an abundance of maternal nucleic acid in the initial biological sample, there is sample degradation, or there is a maternal condition (e.g. , autoimmune disease, transplant rejection, cancer) that increases the amount of maternal oligonucleosomes.
  • Spacer arms may be introduced between the solid support and 5' adapter to improve ligation of the target nucleic acid to the adapter molecule.
  • the ligated sample bound to a solid support may be combined with a ligated sample that does not have a solid support prior to the amplification step.
  • spacer arms refers to any molecule that can be used in single or multiples to create space between the solid support and an oligonucleotide (e.g., target nucleic acid).
  • oligonucleotide e.g., target nucleic acid
  • One or more hexathylene (HEG) spacer units may be inserted between the aminohexyl groups and the 5' end of the 5' adapter. The aminohexyl group is used for covalent coupling to the solid support.
  • HOG hexathylene
  • Other examples of spacer arms that may be used herein include multiple dTTP's (up to 15), spacer 18 (an 18 atom hexa-ethylene glycol spacer), spacer 9 (a triethylene glycol spacer), or photocleavable spacers known in the art.
  • An alternative method disclosed herein selectively detects and amplifies target nucleic acid using a combination of the following 3 steps:
  • any combination of these 3 steps will selectively enrich the double-stranded, blunt-ended 5'-phosphorylated DNA such as DNA from apoptotic ladders by several orders of magnitude over the DNA fragments present in the biological sample that cannot by circularized due to lack of blunt ends and/or missing 5'-terminal phosphate groups and allow a comparison of its sequence with the wild-type sequence of the same organism or the host organism in case of a transplant or a maternal sequence in case of a pregnancy, for instance.
  • step 2 an aliquot of the total DNA is either treated with methylation-sensitive or methylation-resistant enzymes or with chemicals that convert methylated bases into different bases so that methylated bases in the apoptotic DNA fragments can be characterized after step 2 and 3.
  • Circulating nucleic acids in the plasma and serum of patients are associated with certain diseases and conditions (See, Lo YMD et al., N Eng J Med 1998;339:1734-8 ; Chen XQ, et al., Nat Med 1996;2:1033-5 , Nawroz H et al., Nat Med 1996;2:1035-7 ; Lo YMD et al., Lancet 1998;351:1329-30 ; Lo YMD, et al., Clin Chem 2000;46:319-23 ). Further, the method of nucleic acid isolation may affect the ability to detect these disease-associated nucleic acids circulating in the blood ( Wang et al. Clin Chem. 2004 Jan;50(1):211-3 ).
  • the characteristics and biological origin of circulating nucleic acids are not completely understood. However, it is likely that cell death, including apoptosis, is one major factor ( Fournie e al., Gerontology 1993;39:215-21 ; Fournie et al., Cancer Lett 1995;91:221-7 ). Without being bound by theory, as cells undergoing apoptosis dispose nucleic acids into apoptotic bodies, it is possible that at least part of the circulating nucleic acids in the plasma or serum of human subjects is short, fragmented DNA that takes the form particle-associated nucleosomes.
  • the present disclosure provides methods for extracting the short, fragmented circulating nucleic acids present in the plasma or serum of subjects, thereby enriching the short, predictive nucleic acids relative to the background genomic DNA.
  • the present specification provides methods of evaluating a disease condition in a patient suspected of suffering or known to suffer from the disease condition.
  • the specification describes obtaining a biological sample from the patient suspected of suffering or known to suffer from a disease condition, selectively extracting and enriching extracellular nucleic acid in the sample based on its size using the methods provided herein, and evaluating the disease condition by determining the amount or concentration or characteristic of enriched extracellular nucleic acid and comparing the amount or concentration or characteristic of enriched extracellular nucleic acid to a control (e.g., background genomic DNA from biological sample).
  • a control e.g., background genomic DNA from biological sample.
  • evaluating a disease condition refers to assessing the disease condition of a patient.
  • evaluating the condition of a patient can include detecting the presence or absence of the disease in the patient. Once the presence of disease in the patient is detected, evaluating the disease condition of the patient may include determining the severity of disease in the patient. It may further include using that determination to make a disease prognosis, e.g. a prognosis or treatment plan. Evaluating the condition of a patient may also include determining if a patient has a disease or has suffered from a disease condition in the past. Evaluating the disease condition in that instant might also include determining the probability of reoccurrence of the disease condition or monitoring the reoccurrence in a patient.
  • Evaluating the disease condition might also include monitoring a patient for signs of disease. Evaluating a disease condition therefore includes detecting, diagnosing, or monitoring a disease condition in a patient as well as determining a patient prognosis or treatment plan. The method of evaluating a disease condition aids in risk stratification.
  • nucleic acids including DNA and RNA
  • RNA RNA
  • the methods provided herein may be used to extract oncogenic nucleic acid, which may be further used for the detection, diagnosis or prognosis of a cancer-related disorder.
  • nucleic acids including DNA and RNA
  • RNA RNA
  • These molecules are likely packaged in apoptotic bodies and, hence, rendered more stable compared to 'free RNA' ( Anker P and Stroun M, Clin Chem (2002) 48, 1210-1211 ; Ng EK, et al. Proc Natl Acad Sci USA (2003) 100,4748-4753 ).
  • Tumor-specific DNA for a wide range of malignancies has been found: haematological, colorectal, pancreatic, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervix.
  • haematological, colorectal, pancreatic, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervix In aggregate, the above data show that tumor-derived DNA in plasma is ubiquitous in affected patients, and likely the result of a common biological process such as apoptosis. Investigations into the size of these plasma DNA fragments from cancer patients has revealed that the majority show lengths in multiples of nucleosomal DNA, a characteristic of apoptotic DNA fragmentation ( Jahr S, et al. Cancer Res (2001) 61,1659-1665 ).
  • PCR-specific strategies can be developed.
  • clinical application awaits optimization of methods to isolate, quantify and characterize the tumor-specific DNA compared to the patient's normal DNA, which is also present in plasma. Therefore, understanding the molecular structure and dynamics of DNA in plasma of normal individuals is necessary to achieve further advancement in this field.
  • the present specification describes detection of specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic, premalignant or proliferative disease. Specifically, the specification relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA, and to those methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated DNA found in the plasma or serum fraction of blood by using DNA extraction with enrichment for mutant DNA as provided herein.
  • the specification relates to the detection, identification, or monitoring of the existence, progression or clinical status of benign, premalignant, or malignant neoplasms in humans or other animals that contain a mutation that is associated with the neoplasm through the size selective enrichment methods provided herein, and subsequent detection of the mutated nucleic acid of the neoplasm in the enriched DNA.
  • the present specification features methods for identifying DNA originating from a tumor in a biological sample. These methods may be used to differentiate or detect tumor-derived DNA in the form of apoptotic bodies or nucleosomes in a biological sample. In preferred cases, the non-cancerous DNA and tumor-derived DNA are differentiated by observing nucleic acid size differences, wherein low base pair DNA is associated with cancer.
  • the present specification features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample.
  • the DNA species can be differentiated by observing nucleic acid size differences.
  • the differentiation between maternal and fetal DNA may be performed with or without quantifying the concentration of fetal DNA in maternal plasma or serum. Where the fetal DNA is quantified, the measured concentration may be used to predict, monitor or diagnose or prognosticate a pregnancy-associated disorder.
  • non-invasive and invasive techniques available for prenatal diagnosis including ultrasonography, amniocentesis, chorionic villi sampling (CVS), fetal blood cells in maternal blood, maternal serum alpha-fetoprotein, maternal serum beta-HCG, and maternal serum estriol.
  • CVS chorionic villi sampling
  • fetal blood cells in maternal blood maternal serum alpha-fetoprotein
  • maternal serum beta-HCG maternal serum alpha-fetoprotein
  • maternal serum estriol maternal serum estriol
  • the techniques that are non-invasive are less specific, and the techniques with high specificity and high sensitivity are highly invasive.
  • most techniques can be applied only during specific time periods during pregnancy for greatest utility
  • the first marker that was developed for fetal DNA detection in maternal plasma was the Y chromosome, which is present in male fetuses ( Lo et al. Am J Hum Genet (1998) 62:768-775 ).
  • Maternal plasma DNA analysis is also useful for the noninvasive prenatal determination of fetal RhD blood group status in RhD-negative pregnant women ( Lo et al. (1998) N Engl J Med 339:1734-1738 ). This approach has been shown by many groups to be accurate, and has been introduced as a routine service by the British National Blood Service since 2001 ( Finning KM, et al. (2002) Transfusion 42:1079-1085 ).
  • fetal DNA in maternal plasma include the detection of achondroplasia ( Saito H, et al. (2000) Lancet 356:1170 ), myotonic dystrophy ( Amicucci P, et al. (2000) Clin Chem 46:301-302 ), cystic fibrosis ( Gonzalez-Gonzalez MC, et al. (2002) Prenat Diagn 22:946-948 ), Huntington disease ( Gonzalez-Gonzalez MC, et al. (2003) Prenat Diagn 23:232-234 ), and congenital adrenal hyperplasia ( Rijnders RJ, et al. (2001) Obstet Gynecol 98:374-378 ). It is expected that the spectrum of such applications will increase over the next few years.
  • the patient is pregnant and the method of evaluating a disease or physiological condition in the patient or her fetus aids in the detection, monitoring, prognosis or treatment of the patient or her fetus. More specifically, the present specification features methods of detecting abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. The methods according to the present specification provide for detecting fetal DNA in a maternal sample by differentiating the fetal DNA from the maternal DNA based on DNA characteristics ( e.g. , size, weight, 5' phosphorylated, blunt end). See Chan et al. Clin Chem. 2004 Jan;50(1):88-92 ; and Li et al. Clin Chem.
  • fetal DNA that is predictive of a genetic anomaly or genetic-based disease may be identified thereby providing methods for prenatal diagnosis. These methods are applicable to any and all pregnancy-associated conditions for which nucleic acid changes, mutations or other characteristics (e.g. , methylation state) are associated with a disease state. Exemplary diseases that may be diagnosed include, for example, preeclampsia, preterm labor, hyperemesis gravidarum, ectopic pregnancy, fetal chromosomal aneuploidy (such as trisomy 18, 21, or 13), and intrauterine growth retardation.
  • compositions, methods and kits of the present disclosure allow for the analysis of fetal genetic traits including those involved in chromosomal aberrations (e.g. aneuploidies or chromosomal aberrations associated with Down's syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies). Size-based extraction of extracellular fetal DNA in the maternal circulation thus facilitates the non-invasive detection of fetal genetic traits, including paternally inherited polymorphisms which permit paternity testing.
  • chromosomal aberrations e.g. aneuploidies or chromosomal aberrations associated with Down's syndrome
  • genetic markers associated therewith e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies.
  • pregnancy-associated disorder refers to any condition or disease that may affect a pregnant woman, the fetus the woman is carrying, or both the woman and the fetus. Such a condition or disease may manifest its symptoms during a limited time period, e.g., during pregnancy or delivery, or may last the entire life span of the fetus following its birth.
  • a pregnancy-associated disorder include ectopic pregnancy, preeclampsia, preterm labor, and fetal chromosomal abnormalities such as trisomy 13, 18, or 21.
  • chromosomal abnormality refers to a deviation between the structure of the subject chromosome and a normal homologous chromosome.
  • normal refers to the predominate karyotype or banding pattern found in healthy individuals of a particular species.
  • a chromosomal abnormality can be numerical or structural, and includes but is not limited to aneuploidy, polyploidy, inversion, a trisomy, a monosomy, duplication, deletion, deletion of a part of a chromosome, addition, addition of a part of chromosome, insertion, a fragment of a chromosome, a region of a chromosome, chromosomal rearrangement, and translocation.
  • a chromosomal abnormality can be correlated with presence of a pathological condition or with a predisposition to develop a pathological condition.
  • the present specification also provides a method of evaluating the disease condition of a patient suspected of having suffered from a trauma or known to have suffered from a trauma.
  • the method includes obtaining a sample of plasma or serum from the patient suspected of having suffered from a trauma or known to have had suffered from a trauma, and detecting the quantity or concentration of mitochondrial nucleic acid in the sample.
  • the example provides a procedure, using a method provided herein, to selectively extract DNA based on its size.
  • chaotropic salt for example, less than 30% solution (weight per volume) to the sample solution to denature proteins and inactivate nucleases, proteinase K or any protease, for example 100 to 1000 ⁇ g) to further inactivate nucleases and break down proteins in solution.
  • detergents for example SDS or Triton-X 100 up to 1% volume per volume, may be used alone or in combination with a salt.
  • wash target nucleic acid-bound solid support using an appropriate washing solution comprised of salt, buffer, water and alcohol.
  • an appropriate washing solution comprised of salt, buffer, water and alcohol.
  • Figure 1 shows the successful extraction of low base pair DNA from a 1kb DNA ladder (Promega TM ) in the presence of guanidine thiocyanate (GuSCN).
  • the DNA is first bound to silica at various low concentrations of guanidine thiocyanate as shown in Figure 1 .
  • the supernatant from the first binding solution is subsequently bound to silica at varying guanidine thiocyanate concentrations, with a finishing high concentration of 4.6M. These steps are followed by wash and elution steps.
  • the method can be employed to produce size selective separation of a commercially available DNA ladder with DNA strands ranging in mass from 250bp to 10,000 bp from normal human plasma in the presence of guanidine thiocyanate as the chaotropic salt. The following steps are performed:
  • Figure 2 shows the successful extraction of low base pair DNA from a 1kb DNA ladder (Promega TM ) in the presence of sodium perchlorate (NaClO 4 ).
  • the DNA is first bound to silica at various low concentrations of sodium perchlorate as shown in Figure 2 .
  • the supernatant from the first binding solution is subsequently bound to silica at varying sodium perchlorate concentrations, with a finishing high concentration of 4.5 M. These steps are followed by wash and elution steps.
  • the method can be employed to produce size selective separation of a commercially available DNA ladder with DNA strands ranging in mass from 250bp to 10,000 bp from normal human plasma in the presence of sodium perchlorate (NaClO 4 ) as the chaotropic salt.
  • NaClO 4 sodium perchlorate
  • the below example provides a procedure, using the method provided herein, to selectively amplify target nucleic acid that is blunt-ended and 5'-phosphorylated.
  • the method relies on the ligation of a non-genome specific adapter to the blunt ends, which allows for whole genome amplification followed by target-specific amplification. While the nature of the termini of all cell-free nucleic acid is unknown, coupling this method with short extension times during amplification will favor the amplification of the oligonucleosome monomer and short multimers. Since the target nucleic acid is shorter than the non-targeted nucleic acid, the target nucleic acid can be enriched over the non-target nucleic acid. This method can be further coupled with specific amplification of a nucleic acid region of interest for further analysis. Figure 3 shows results of the method.
  • the 3' adapter is complementary to the 3'-end of 5' adapter to create the blunt-end, double-stranded adapter complex.
  • 5' adapter 5'-ACACGGCGCACGCCTCCACG-3' ⁇ blunt-end
  • 3' adapter 3'-GTGCGGAGGTGC-5'
  • the 3' adapter molecule is modified such that the new sequence consists of 13 nucleobases with a dideoxy-nucleotide at its 3'-position.
  • This terminator nucleotide does not allow extension of the 3' adapter molecule by any polymerase, thus improving assay efficiency and detection.
  • the 3' adapter is shorter to reduce the melting temperature and allow for release from the 5' adapter following ligation.
  • Both adapters are non-phosphorylated, and may be made of any sequence that is nonspecific to the nucleic acid to be amplified to prevent non-specific amplification of the genome.
  • An exemplary procedure is provided below: 1. Prepare total or size selective nucleic acid sample in water or buffer. 2. Add ligation buffer, 5' adapter, 3' adapter, and water to reaction volume. 3. Place the reaction into a thermocycler and heat the reaction to 55°C for 10 minutes, then slowly ramp down the temperature to 10°C over 1 hour. 4. Add 1 ⁇ l T4 ligase (1-3 U per ul) (or ligation enzyme) and mix well and incubate for 10 min at 10°C, then ramp temperature up to 16°C and incubate for 10 minutes to over night (12-16 hours). 5. Add 5' primer, 10x PCR buffer, MgCl 2 , dNTPs, and polymerase. 6.
  • Described hereafter is a method for intramolecular ligation followed by amplification of a target by inverse PCR or rolling circle amplification to detect a target nucleic acid.

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Claims (4)

  1. Kit zum Extrahieren von Zielnukleinsäure, umfassend:
    a) eine wässerige Lösung von paramagnetischen Mikropartikeln mit einer beschichteten Oberfläche aus funktionellen Gruppen;
    b) einen ersten Bindepuffer, dergestalt formuliert, dass er ein geeignetes Salz enthält, wobei das Salz in einer Konzentration vorhanden ist, die für die Bindung von Nichtzielnukleinsäuren von 1200 oder mehr Basenpaaren an den paramagnetischen Mikropartikeln geeignet ist; und
    c) einen zweiten Bindepuffer, dergestalt formuliert, dass er ein geeignetes Salz enthält, wobei das Salz in einer höheren Konzentration als in dem ersten Bindepuffer vorliegt und für die Bindung einer Zielnukleinsäure von weniger als 1200 Basenpaaren an den paramagnetischen Mikropartikeln geeignet ist, wobei das geeignete Salz in dem ersten Bindepuffer und in dem zweiten Bindepuffer mindestens eine Verbindung ausgewählt aus der Gruppe bestehend aus Guanidinsalz, Natriumiodid, Natriumperchlorat, Kaliumiodid, Natriumthiocyanat, Harnstoff, Natriumchlorid, Magnesiumchlorid, Calciumchlorid, Kaliumchlorid, Lithiumchlorid, Bariumchlorid, Cäsiumchlorid, Ammoniumacetat, Natriumacetat und Ammoniumperchlorat ist.
  2. Kit nach Anspruch 1, wobei das Kit Reagenzien für die Formulierung eines Waschpuffers und eines Elutionspuffers umfasst, wobei der Waschpuffer Verunreinigungen auflöst, aber nicht die Nukleinsäure, die an den paramagnetischen Mikropartikeln gebunden ist, und der Elutionspuffer eine nicht-salzige Pufferlösung mit einem pH-Bereich von etwa 7,0 bis etwa 8,5 ist.
  3. Kit nach Anspruch 1 oder 2, wobei die mit funktionellen Gruppen beschichtete Oberfläche der paramagnetischen Mikropartikel Silica-beschichtet, hydroxylbeschichtet, aminbeschichtet, carboxylbeschichtet oder mit verkapselten Carboxylgruppen beschichtet ist.
  4. Kit nach einem der Ansprüche 1 bis 3, wobei das Salz Guanidin-(iso)thiocyanat oder Natriumperchlorat ist.
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